The present invention relates generally to the field of product identification; and more specifically, to the application of biotechnological systems to mark products for identification.
The interaction between a variety of ligands and the receptors with whiche they bind intracellularly has been exploited in fields where the triggering of a receptor-induced promoter enables the promoter-regulated expression of a gene encoding a desired protein. Such inducible expression systems permit the desired protein to be produced in the cell at an appropriate timepoint. One such receptor system is the insect steroid hormone receptor system disclosed in U.S. Pat. No. 5,514,578.
Such systems are used in drug or new compound screening. For example, International patent application No. WO092/27356, published Jun. 3, 1999, refers to methods for identifying modulators of nuclear hormone receptor function by mixing the receptor, a peptide sensor, and a test compound. The sensor provides direct binding to the receptor, and an assay is performed to determine if the test compound influenced the binding of the sensor protein to the receptor. Additionally, Evans, U.S. Pat. No. 5,071,773 refers to hormone receptor related bioassays as screens to determine whether proteins are receptors that activate transcription or whether a test agent is a ligand that activates a known receptor.
Such receptor systems have been proposed for xe2x80x9cfingerprintingxe2x80x9d or as biosensors for marking products for identification. For example, the interaction between certain G-protein coupled cell surface receptors, tyrosine canasta receptors, and ion channel receptors which have been mutated to have altered binding to their natural ligands and various non-natural ligands thereto, have been proposed for the generation of a sample fingerprint. The fingerprint is proposed to enable the authentification and monitoring of products for safety, security, fraud and quality control. See, International Patent Application No. W099/51777, published Oct. 14, 1999; and International Patent Application No. W097/15985, published Oct. 2, 1997. International Patent Application No. W095/0282, published Jan. 26, 1995 refers to a method of detecting a ligand by incubating cells transfected with DNA coding for a receptor that can influence cell amplification in response to the ligand, with a test substance that is a potential agonist or antagonist of the receptor. A marker for amplification in the cells is then used to assess the presence or absence of amplification of cells.
Some disadvantages of these prior art detection systems include a need for light concentration of ligand in the marked product as well as a limitation on the number and character of the ligands that can be used in the detection method.
There is a need in the art for additional uses of receptor-ligand interactions for product identification which interactions can generate simple and rapid signals at low concentrations.
In one aspect, the present invention provides an improved method for identifying a product, which employs ligand-dependent transcription factors. This method involves first associating a marker ligand with the product, and then detecting the marker ligand in the product or a portion or extract thereof at a later point in time as a means of identifying the product. The ligand-containing product is contacted with a detector composition comprising one or more first nucleotide sequences encoding one or more natural or synthetic ligand- dependent transcription factors. The ligand-dependent transcription factor(s) comprise at least one ligand binding domain, at least one DNA binding domain and at least one transactivation domain. The factor(s) are preferably under the regulatory control of a first promoter. The detector composition also comprises a second nucleotide sequence encoding a reporter gene under the regulatory control of a receptor response element or a modified or synthetic response element, and a second promoter. The interaction between the marker ligand and at least one of the ligand binding domains is highly specific and induces a change in the expression of the reporter gene. This change produces a detectable signal identifying the presence of the ligand in the product.
In another aspect, the invention provides an additional method such as that described above, but wherein the detector composition further comprises a third nucleotidc sequence encoding a coactivator or corepressor that interacts with the ligand-dependent transcription factor to activate or repress expression of said reporter gene. In still another embodiment of a method of this invention, the product is contacted with the detector composition and either a coactivator or repressor protein that interacts with the ligand-dependent transcription factor to activate or repress expression of said reporter gene.
In another aspect, the invention provides a marked product comprising a liquid, a solid, a dispersion, an emulsion, or a latex associated with a specified marker ligand described in detail below.
In a further aspect, the invention provides one or more cells or a stable cell line comprising the above-described first nucleotide sequence(s) and the above-described second nucleotide sequence, and optionally the coactivator or corepressor for use in the claimed method.
In still another aspect, the invention provides a kit for identifying a marked product comprising a detector composition comprising the above-described first and second nucleotide sequences, and means for detecting and measuring the signal.
In yet another aspect, the invention provides detector compositions for such use.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.